Cloning and regulation of an endothelial cell protein C/activated protein C receptor

ABSTRACT

Human protein C and activated protein C were shown to bind to endothelium specifically, selectively and saturably (Kd=30 nM, 7000 sites per cell) in a Ca 2+  dependent fashion. Expression cloning revealed a 1.3 kb cDNA that coded for a novel type I transmembrane glycoprotein capable of binding protein C. This protein appears to be a member of the CD1/MHC superfamily. Like thrombomodulin, the receptor involved in protein C activation, the endothelial cell protein C receptor (EPCR) function and message are both down regulated by exposure of endothelium to TNF. Identification of EPCR as a member of the CD1/MHC superfamily provides insights into the role of protein C in regulating the inflammatory response, and determination of methods for pharmaceutical use in manipulating the inflammatory response.

This application is a divisional of U.S. Ser. No. 09/182,616, filed onOct. 29, 1998, now U.S. Pat. No. 6,399,064, which is a divisional ofU.S. Ser. No. 08/878,283, filed on Jun. 18, 1997, now U.S. Pat. No.5,852,171, which is a divisional of U.S. Ser. No. 08/289,699, filed onAug. 12, 1994, now U.S. Pat. No. 5,695,993.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of cloning, expression,and regulation of an endothelial cell protein C/activated protein Creceptor.

Protein C plays a major role in the regulation of blood coagulation.Patients deficient in protein C usually exhibit life threateningthrombotic-complications in infancy (Seli{overscore (g)}sohn et al.,(1984) N. Engl. J. Med. 310, 559–562; Esmon, (1992) Trends Cardiovasc.Med. 2, 214–220) that are corrected by protein C administration (Dreyfuset al., (1991) N. Engl. J. Med. 325, 1565–1568). In addition, activatedprotein C (APC) can prevent the lethal effects of E. coli in baboonmodels of gram negative sepsis (Taylor et al., (1987) J. Clin. Invest.79; U.S. Pat. No. 5,009,889 to Taylor and Esmon) and preliminaryclinical results suggest that protein C is effective in treating certainforms of human septic shock (Gerson et al., (1993) Pediatrics 91,418–422). These results suggest that protein C may both controlcoagulation and influence inflammation. Indeed, inhibition of protein S,an important component of the protein C pathway, exacerbates theresponse of primates to sublethal levels of E. coli and augments theappearance of TNF in the circulation (Taylor et al., (1991) Blood 78,357–363). The mechanisms involved in controlling the inflammatoryresponse remain unknown.

Protein C is activated when thrombin, the terminal enzyme of thecoagulation system, binds to an endothelial cell surface protein,thrombomodulin (Esmon, (1989) J. Biol. Chem. 264, 4743–4746; Dittman andMajerus, (1990) Blood 75, 329–336; Dittman, (1991) Trends Cardiovasc.Med. 1, 331–336). In cell culture, thrombomodulin transcription isblocked by exposure of endothelial cells to tumor necrosis factor (TNF)(Conway and Rosenberg, (1988) Mol. Cell. Biol. 8, 5588–5592) andthrombomodulin activity and antigen are subsequently internalized anddegraded (Lentz et al., (1991) Blood 77, 543–550, Moore, K. L., et. al.,(1989) Blood 73, 159–165). In addition, C4bBP, a regulatory protein ofthe complement system, binds protein S to form a complex that isfunctionally inactive in supporting APC anticoagulant activity in vitro(Dahlbäck, (1986) J. Biol. Chem. 261, 12022–12027) and in vivo (Taylor,et al., 1991). Furthermore, C4bBP behaves as an acute phase reactant(Dahlbäck, (1991) Thromb. Haemostas. 66, 49–61). Thus, proteins of thispathway not only appear to regulate inflammation, but they also interactwith components that regulate inflammation, and they themselves aresubject to down regulation by inflammatory mediators.

Given the central role of the protein C pathway in regulating the hostresponse to inflammation and the critical role of the pathway incontrolling blood coagulation, it is important to identify andcharacterize all of the components that interact with the system. Thisis especially true since the molecular basis of the anti-inflammatoryeffects of the protein C pathway components have yet to be elucidated atthe molecular level.

It is therefore an object of the present invention to provide a cellularreceptor for protein C and activated protein C.

It is a further object of the present invention to provide nucleotidesequences encoding the cellular receptor and amino acid characterizationof the receptor which allows expression of recombinant native andmodified forms of the receptor.

It is another object of the present invention to provide methods ofmodulating the inflammatory response involving protein C and activatedprotein C.

SUMMARY OF THE INVENTION

An endothelial cell protein C binding protein (referred to herein as“EPCR”) has been cloned and characterized. The protein is predicted toconsist of 238 amino acids, which includes a 15 amino acid signalsequence at the N-terminus, and a 23 amino acid transmembrane regionwhich characterizes the receptor as a type 1 transmembrane protein. Theprotein binds with high affinity to both protein C and activated proteinC (Kd=30 nM) and is calcium dependent. The message and binding functionof the receptor are both down regulated by cytokines such as TNF.

These results identify a new member of a complex pathway that, likeother members of the pathway, is subject to regulation by inflammatorycytokines, and can therefore be used to modulate inflammatory reactionsin which protein C or activated protein C is involved. Inhibition of theinflammatory response can be obtained by infusing soluble EPCR.Alternatively, localizing EPCR to surfaces in contact with blood willrender the surfaces anticoagulant by virtue of the ability of EPCR tobind and concentrate the anticoagulant activated protein C at thesurface. Alternatively, the function of EPCR can be enhanced byoverexpressing the EPCR in endothelium that could be used to coatvascular grafts in patients with vascular disease or on stents incardiac patients.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B and 1C are flow cytometric analyses of F1-APC (fluorescentlabelled activated protein C) binding to HUVEC (human umbilical veinendothelial cells). FIG. 1A is a graph of cell number versus log offluorescence intensity, demonstrating F1-APC binding to HUVEC. HUVEC(1×10⁵) were incubated at room temperature without (dotted line) or with160 nM of F1-APC (solid line) in the presence of 1.3 mM CaCl₂. Afterwashing, bound APC was analyzed by flow cytometry. FIG. 1B is a graph offluorescence intensity versus F1-APC concentration (nM) demonstratingthe concentration dependence of F1-APC binding to HUVEC. HUVEC wereincubated with F1-APC in the absence (open circles) or presence of 1.3mM CaCl₂ (closed circles) and binding was measured as in A. Mean channelfluorescence intensity is plotted for each F1-APC concentration (between0 and 800 nM). FIG. 1C is a graph of the percent of mean fluorescenceversus unlabeled protein concentration (μg/ml), demonstrating theeffects of unlabeled proteins on F1-APC binding to HUVEC. F1-APC bindingto HUVEC was carried out in the presence of the indicated concentrations(between 0 and 100 μg/ml) of unlabeled APC, protein C, protein S, factorX and Xa or recombinant Gla-domainless protein C (rGDPC).

FIGS. 2A, 2B, 2C and 2D are graphs of ¹²⁵I-APC Binding to HUVECMonolayers. FIG. 2A is a graph of the bound APC (cpm×10⁻³) versus time(min), showing the time course of ¹²⁵I-APC binding to HUVEC. HUVECmonolayers (1.2×10⁵ cells) were incubated at 4° C. with 32 nM (filledsquares) or 8 nM (open squares) I¹²⁵I-APC. At the indicated times, cellswere washed and bound radioactivity was measured. FIG. 2B is a graph ofbound APC (cpm×10⁻³) versus unlabeled protein (nM) demonstrating theeffects of unlabeled APC and rGDPC on ¹²⁵I-APC binding to HUVEC. HUVECwere incubated at 4° C. for one hour with ¹²⁵I-labeled APC in thepresence of the indicated concentrations (between 01 and approximately1000 nM) of unlabeled APC (open circles) or rGDPC (closed circles).After washing, bound radioactivity was measured. FIG. 2C is a graph ofbound APC (fmol/well) versus free APC (nM) demonstrating theconcentration dependence of ¹²⁵I-APC binding to HUVEC. Monolayers ofHUVEC were incubated with the concentrations of ¹²⁵I-APC indicated asdescribed above. Specific binding was determined as described below.FIG. 2D is a Scatchard analysis of ¹²⁵I-APC binding to HUVEC. Each valuewas calculated from the data shown in FIG. 2C.

FIGS. 3A and 3B are flow cytometric analyses of F1-APC binding to 293Tcells transfected with a cDNA clone of EPCR. Cells were transfected witha clone EPCR/pEF-BOS or pEF-BOS (negative control) by thecalcium/phosphate method. After 24 h, cells were harvested and F1-APCbinding was performed in the absence (dotted lines) or presence of 1.3mM CaCl2 (solid lines).

FIG. 4 is the predicted protein structure of EPCR based on nucleotidesequence (SEQ ID NO:1) predicted amino acid sequence (SEQ ID NO:2) ahydropathy plot of EPCR. The signal sequence and transmembrane regionare indicated with the solid bars.

FIG. 5 is a comparison of the amino acid sequence of EPCR to the aminoacid sequences of other members of the CD1 family and CCD41. The EPCRsequence (SEQ ID NO:2) is shown in the first line and compared to murineCCD41 (SEQ ID NO:3) (second line) human CD1d (SEQ ID NO:4) (third line)and murine CD1.2 (SEQ ID NO:5) (fourth line). Identities with EPCR areindicated by open boxes. Residues that are conserved between EPCR andall of the human CD1 family members are indicated by a double asterisk.Residues shared with one or more members of the CD1 family are indicatedby a single asterisk.

FIG. 6 is a comparison of the amino acid sequence of human EPCR (SEQ IDNO:2) (first line) to the amino acid sequence of murine EPCR (SEQ IDNO:6) (second line). Identities are indicated by boxes. Similarities areindicated with an asterisk.

DETAILED DESCRIPTION OF THE INVENTION

I. Cloning and Characterization of EPCR.

Human protein C and activated protein C are shown to bind to endotheliumspecifically, selectively and saturably (Kd=30 nM, 7000 sites per cell)in a Ca²⁺ dependent fashion. FL-APC binding to various human cell lineswere examined, and found that the binding was HUVEC specific. A humankidney cell line transformed with SV40 large T antigen, 293T cells,expressed very few of these binding sites. A HUVEC cDNA library wasconstructed using the powerful mammalian expression vector, pEF-BOS(Mizushima and Nagata, (1990) Nucleic Acids Res. 18, 5322). Plasmid DNAwas prepared from subpools of independent colonies (2,500 colonies perpool), and transfected into 293T cells, using the method of Kaisho etal., (1994) Proc. Natl. Acad. Sci. (USA) 91, 5325. FL-APC binding wasanalyzed on a flow cytometer. One of eight subpools gave a positivesignal. This subpool was divided into 20 subpools and rescreened. Afterthree rounds of enrichment, one positive clone, EPCR-1, was isolated.EPCR-1 carries a 1.3 kb insert. When transfected into 293T cells, thisclone was capable of expressing the calcium-dependent binding site forFL-APC on the 293T cell surface.

Expression cloning revealed a 1.3 kb cDNA that coded for a type Itransmembrane glyceprotein capable of binding protein C. This proteinappears to be a member of the CD1/MHC superfamily. Like thrombomodulin,the receptor involved in protein C activation, the endothelial cellprotein C receptor (EPCR) function and message are both down regulatedby exposure of endothelium to TNF. Identification of EPCR as a member ofthe CD1/MHC superfamily provides insights into the role of this receptorfor protein C in regulating the inflammatory response.

Materials and Methods

Protein Preparation

Human protein C (Esmon et al., (1993) Meths. Enzymol. 222, 359–385), APC(Esmon et al., 1993), recombinant gla domainless protein C (rGDPC)(Rezaie et al., (1992) J. Biol. Chem. 267, 11701–11704), protein S(Taylor et al., 1991), factor X and factor Xa (Le Bonniec et al., (1992)J. Biol. Chem. 267, 6970–6976) were prepared as described in the citedpublications.

Selective labeling of the active site of APC with fluorescein wasperformed by the method of Bock (Bock, P. E. (1988) Biochemistry 27,6633–6639). In brief, N^(α)[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH₂C1 (200μM) was reacted with 40 μM APC for 1 hour at room temperature. Afterdialysis, the covalently modified APC was incubated at room temperaturefor one hour with 200 μM 5-(iodoacetamido)fluorescein (MolecularProbes). Free fluorescein was removed by gel filtration on a PD-10column (Pharmacia). With this method, each molecule of fluoresceinatedAPC (FI-APC) contains a single dye at the active site and hence all ofthe fluorescent molecules behave identically.

Iodogen (Pierce) was used to radiolabel APC with Na[¹²⁵I] (Amersham)according to the manufacture's protocol in the presence of 5 mM CaCl₂.Free ¹²⁵I was removed by gel filtration on a PD-10 column. The specificactivity of the ¹²⁵I-APC was 1×10⁴ cpm/ng protein.

Cell Culture

Human umbilical vein endothelial cells (HUVEC) were isolated from freshumbilical-cords by collagenase treatment and cultured in medium 199containing 15% fetal bovine serum, 10 μg/ml heparin, and 0.5%endothelial cell growth supplement prepared from bovine brain extract(Maciag at al., (1979) Proc. Natl. Acad. Sci. (USA) 76, 5674–5678). HOS(ATCC CRL 1543), HEp-2 (ATCC CCL 23) and 293 cells (ATCC CRL 1573)transformed with SV40 large T antigen (293T, a gift from Dr. KenjiOritani) were maintained in Earl's MEM supplemented with 10% fetalbovine serum. The human lymphocyte cell lines, Jurkat, MOLT3 (ATCC CRL1552), Jijoye (ATCC CCL 87), Raji (ATCC CCL 86), U-937 (ATCC CRL 1593),HL-60 (ATCC CCL 240), and HEL (ATCC TIB 180), were maintained inRPMI-1640 medium supplemented with 10% fetal bovine serum.

Flow Cytometric Analysis of F1-APC Binding to Cells

Adherent cells were harvested by incubation at 37° C. for 5 min inphosphate buffered saline (PBS) containing 0.02% EDTA. Cells were washedtwice with EDTA/PBS and then once with Hank's balanced salt solution(HBSS). They were resuspended in HBSS containing 1% bovine serum albumin(BSA) and 0.02% sodium azide (binding buffer). Cells (1×10⁵) wereincubated at room temperature for 45 min with F1-APC in the dark. Afterwashing, they were resuspended in the binding buffer containing 0.5μg/ml of propidium iodide. Bound F1-APC was analyzed on a flowcytometer, FACScan (Becton Dickinson). Living cells were gated on a dotplot display of forward-scatter (FSC) versus fluorescence-2 (FL2), andF1-APC binding was detected on the fluorescence-1 (FL-1) channel. Allexperiments were performed in duplicate.

¹²⁵I-APC Binding to HUVEC

Monolayers of HUVEC in 24-well microplates (Costar) (1×10⁵ cells perwell) were washed twice with EDTA/PBS and once with ice-cold HBSS. Cellswere then incubated at 4° C. for one hour in the binding buffer with¹²⁵I-APC. After washing three times with ice-cold HBSS, cells werereleased with the EDTA buffer, and the bound radioactivity was measuredin a gamma counter (Isodata 500). To determine non-specific,calcium-independent adsorption of radioactivity, the cells were washedwith EDTA/PBS and residual radioactivity in the cell pellet wasmeasured. Non-specific binding of radioactivity was consistently lessthan 5% of the specific binding. The data was analyzed using theEnzfitter program (Elsevier Biosoft, Cambridge, U.K.).

Construction of HUVEC cDNA Library

Poly-A RNA was isolated from HUVEC (1×10⁸ cells) using the FastTrack™mRNA isolation kit (Invitrogen). cDNA was synthesized from 3 μg ofpoly-A RNA using a Librarian™ I kit (Invitrogen). A BstX I adaptor wasligated, double stranded cDNA was fractionated by agarose gelelectrophoresis, and cDNA longer than 700 bp was ligated into amammalian expression vector, PEF-BOS (Mizushima and Nagata, 1990; thisvector was a kind gift from Dr. S. Nagata). The construct wastransfected into E. coli-DH10B by electroporation (Bio-Rad GenePulser™). The library-consisted of 8×10⁶ independent colonies with anaverage size of 2.0 kb.

Expression Cloning and Sequence Analysis

Approximately 2×10⁴ independent colonies were divided into eightsubpools (each containing 2,500 independent colonies) and plasmid DNAwas prepared from each subpool. Sub-confluent 293T cells in 24-wellmicroplates were transfected with 1 μg of the DNA by thecalcium/phosphate method (Graham and Van Der Eb, (1973) Virology 52,456–467). After 20 hours, the medium was changed, and culture wascontinued for another 24 hours. The subpools were screened for F1-APCbinding by FACS analysis as described above. The positive library poolwas then divided into 20 new pools and rescreened. After three rounds ofscreening, 96 individual clones were tested and one positive clone wasidentified.

The insert (1.3 kb) was subcloned into pBluescript™ (Stratagene), andthe nucleotide sequence was determined using a Sequenase™ version 2.0DNA Sequencing kit (USB). Nucleotide and protein database searchemployed the BLAST™ (NCBI) and FASTA™ programs (GCG) with GenBank, EMBL,and SwissProt databases.

Northern Blot Analysis

Total RNAs (15 μg) from various cells were isolated, electrophoresedthrough formaldehyde agarose gels and transferred to a nylon membrane(Hybond-N™, Amersham). The 483 bp Xba I fragment from the 5′ end of theEPCR cDNA was labeled by random priming according to the manufacturer'sinstructions (Multiprime™ DNA labeling system, Amersham) and used forhybridization.

Protein C and APC Binding to HUVEC

Endothelial cells in suspension bound FL-APC, as monitored by flowcytometry, and demonstrated in FIG. 1A. Binding was saturable and Ca²⁺dependent, as shown by FIG. 1B. Optimal binding required at least 1 mMCa²⁺. FL-APC was displaced from the cell surface by APC and protein Cequivalently, as shown by FIG. 1C. The homologous Gla-domain containingproteins, protein S, factor X, and its active form, factor Xa, failed todisplace bound F1-APC, suggesting that there is a specific binding sitefor APC on the endothelial cell surface. Protein C binding was dependenton the Gla domain, since recombinant gla-domainless protein C (rGDPC)failed to displace F1-APC.

Detailed binding studies were also performed with ¹²⁵I-labeled APC andmonolayers of HUVEC, as shown by FIGS. 2A, 2B, 2C and 2D. The bindinganalysis indicated 7,000 sites per cell and a Kd=30 nM. This affinity issimilar to that estimated from FIG. 1.

Endothelial cell surface thrombomodulin can interact with protein C andAPC. The Kd (greater than 1 μM) (Hogg et al., (1992) J. Biol. Chem. 267,703–706; Olsen et al., (1992) Biochemistry 31, 746–754), however, ismuch higher than that of the binding site described above with respectto the new receptor. Furthermore, polyclonal and monoclonal antibodiesagainst thrombomodulin that inhibit protein C activation did not inhibitthe binding. Protein S also can interact with protein C and APC(Dahlbäck et al., (1992) Biochemistry 31, 12769–12777), but F1-APCbinding to HUVEC was not influenced by protein S addition. Furthermore,polyclonal and monoclonal antibodies to protein S did not inhibit thebinding. These results indicate the binding site for protein C and APCon endothelium is distinct from these known molecules.

Nucleotide and Predicted Protein Structure Analysis of EPCR

The insert was subcloned into pBluescript, and the nucleotide sequencewas determined, as shown in Sequence ID No. 1. The cDNA shown inSequence ID No. 1 consists of 1302 bp, including a translationinitiation ATG codon (AGGATGT, (Kozak, (1986) Cell 44, 283–292) at the5′-end at nucleotides 25–27 of Sequence ID No. 1. A potentialpolyadenylation signal sequence, AATAAA, (Proudfoot and Brownlee, (1976)Nature 263, 211–214) begins at nucleotide 1267 of Sequence ID No. 1,just 18 bp upstream of the poly(A) sequence.

The cDNA is predicted to code for a protein of 238 amino acids (SequenceID No. 2), which includes a 15 amino acid signal sequence (von Heijne,(1986) Nucleic Acids Res. 14, 4683–4690) at the N-terminal. Therefore,the mature protein is predicted to contain 223 amino acids. Sequence IDNo. 2 is the predicted amino acid sequence of EPCR. Amino acids 1–15 ofSequence ID No. 2 (MLTTLLPILLLSGWA) are the putative signal sequencedetermined by the method of von Heijne (von Heijne, 1986). Amino acids211–236 of Sequence ID No. 2 (LVLGVLVGGFIIAGVAVGIFLCTGGR) are theputative transmembrane domain. Potential N-glycosylation sites arepresent at amino acids 47–49, 64–66, 136–138, and 172–174 of Sequence IDNo. 2. Extracellular cysteine residues are present at amino acids 17,114, 118, and 186 of Sequence ID No. 2. A potential transmembrane region(Engelman et al., (1986) Annu. Rev. Biophys. Biophys. Chem. 15, 321–53)consisting of 23 amino acids was identified at the C-terminal end(beginning at amino acid 216 of Sequence ID No. 2).

The protein is predicted to be a type 1 transmembrane protein. Theextracellular domain contains four potential N-glycosylation sites andfour Cys residues. The cytoplasmic region contains only three aminoacids and terminates with a Cys, which could be acylated to something orinvolved in heterodimer formation with another peptide.

Although described with reference to cloning and expression of theprotein encoding sequence, larger amounts of protein can be obtained byexpression in suitable recombinant host systems, such as mammalian,yeast, bacteria, or insect cells. Isolation can be facilitated by makingantibodies to the recombinant protein which are then immobilized onsubstrates for use in purification of additional receptors, as describedbelow.

As used herein, the nucleotide sequences encoding the receptor includethe sequence shown in Sequence ID No. 1, and sequences havingconservative substitutions, additions or deletions thereof whichhybridize to Sequence ID No. 1 under stringent conditions. As usedherein, the amino acids sequences constituting the receptor include thesequence shown in Sequence ID No. 2, and sequences having conservativesubstitutions, additions or deletions thereof which form a receptorhaving functionally equivalent biological activity. It is well known tothose skilled in the art what constitutes conservative substitutions,additions or deletions, and which could be readily ascertained asencoding, or forming, a functionally equivalent receptor molecule usingthe functional assays described herein.

The hydropathic plot shown in FIG. 4 was performed according to themethod of Goldman et al (Engelman et al., 1994) (solid line) and that ofKyte and Doolittle (1982) J. Mol. Biol. 157, 105–132 (dotted line).

DNA and protein database searches revealed that the sequence is relatedto the centrosome-associated, cell cycle dependent murine protein,CCD41, also referred as centrocyclin (Rothbarth et al., (1993) J. CellSci. 104, 19–30), as shown by FIG. 5. The similarity in the publishedsequence of murine CCD41 with human EPCR led to the cloning andsequencing of the murine EPCR. The sequence of murine EPCR is shown inFIG. 6. It is distinct from the published sequence of CCD41.

The EPCR amino acid sequence was also related to, but quite distinctfrom, the CD1/MHC superfamily and the murine CD1.2, as also shown byFIG. 5. Based on the homology to CD1/MHC, it is likely that EPCRcontains two domains consisting of residues 17–114 and 118–188. Of theCD1 family members, CD1d is the most similar to EPCR. In the mouse,CCD41 is associated exclusively with the centrosome during G₁ butbecomes detectable elsewhere during the cell cycle, reaching a maximumduring G₂, except during the G₂/M phase (Rothbarth et al., 1993). EPCRexpression appears restricted to endothelium, which would not beexpected for a cell cycle associated protein.

The identification of the protein C receptor on endothelium suggeststhat the endothelial cell binds protein C/APC through three distinctmechanisms. In addition to EPCR, protein S can bind APC/protein C onnegatively charged membrane surfaces that include the endothelium (Sternet al., (1986) J. Biol. Chem. 261, 713–718), but this is not cell typespecific (Dahlbäck et al., 1992). Thrombomodulin in complex withthrombin can bind protein C and APC (Hogg et al., 1992). On endothelium,the protein S binding sites (Nawroth and Stern, (1986) J. Exp. Med. 163,740–745), thrombomodulin (Esmon, 1989) and EPCR are all down regulatedby cytokines, indicating that inflammation can impair protein C pathwayfunction at multiple levels.

The homology to the CD1/MHC family of proteins is especially interestingsince it provides indications as to the function of EPCR. The CD1/MHCfamily has three extracellular domains termed α1,2 and 3. Theextracellular domain of EPCR contains four Cys residues that appear tocorrespond to two distinct domains. EPCR lacks the third domain of theCD1/MHC family, but the two domains have significant homology to the α1and α2 domains of the CD1 protein family and the α2 domain of the MHCclass 1 protein, suggesting that these proteins evolved from a commonancestor. The first domain of EPCR, residues 17–114, contains twopotential N glycosylation sites and is rich in β strand structure,suggesting that it may form a β sheet. Despite the β strand structure,consensus sequences (Williams and Barclay, (1988) Ann. Rev. Immunol. 6,381–405) for the immunoglobulin superfamily of receptors are absent. Thesecond domain of EPCR, residues 118–188, contains two additional Nglycosylation sites and, like the CD1/MHC family, this domain ispredicted to have limited B structure.

II. Modulation of Inflammation using EPCR.

In vitro studies have suggested anti-inflammatory activities for APC.For instance, an unusual carbohydrate sequence on protein C can inhibitinflammatory cell adhesion to selectins (Grinnell at al., (1994)Glycobiology, 4, 221–226) Modest inhibitory effects of APC have beenreported on TNF production (Grey et al., (1993) Transplant. Proc. 25,2913–2914). EPCR could contribute to these anti-inflammatory mechanisms.Since the homologous protein family, CD1, can be linked to CD8(Ledbetter et al., (1985) J. Immunol. 134, 4250–4254), it is alsopossible that the proteins C receptor is linked to another protein andsignal through this second protein. One of the CD1 family members, CD1d,has been reported to promote T cell responses, possibly involvingbinding to CD8 (Panja et al., (1993) J. Exp. Med. 178, 1115–1119). CD1bhas recently been reported to serve as an antigen presenting molecule(Porcelli et al., (1992) Nature 360, 593–597). The ability to bindprotein C/APC could then be linked either directly or indirectly tosignalling via direct interaction with cells of the immune system. Sincethe MHC class of proteins is involved in presentation of proteins tocell receptors, the concept of presentation of protein C/APC toinflammatory cells as a means of elaborating anti-inflammatory activitymay also be involved. This includes modulation of enzyme specificitysuch as occurs with thrombin-thrombomodulin interaction (Esmon, 1989).In this case, the EPCR-APC complex might cleave biologically activepeptides from unknown substrates.

EPCR mRNA Levels and APC Binding

To determine the cellular specificity of EPCR expression, the intensityof FL-APC binding to HUVEC was compared to several human cell lines.F1-APC bound strongly only to HUVEC, and not to any of the T, B, ormonocytic cell lines tested. Cells were incubated at room temperaturewithout or with 160 nM F1-APC in the presence of 1.3 mM CaCl₂. Bindingwas analyzed by flow cytometry. Slight binding was demonstrated with theosteosarcoma line, HOS and the epidermoid carcinoma cell line, HEp-2.

Total RNA was extracted from these cells and hybridized with the EPCRcDNA probe for Northern Blot Analysis. EPCR mRNA was detected byNorthern blot analysis for HUVEC, Jurkat, HEp-2, Raji, HOS, and U937.Among the cells lines tested, EPCR mRNA was detected at high levels onlyin HUVEC. The calculated mRNA size of 1.3 kb was identical to the sizeof the isolated cDNA. After prolonged exposure, a weak signal was alsodetected with the osteosarcoma cell line HOS and monocyte cell lineU937. Thus, both APC binding and EPCR mRNA expression are very specificfor endothelium.

Effects of TNF on APC Binding and EPCR mRNA Levels

Several other members of the protein C anticoagulant pathway are subjectto regulation by inflammatory cytokines (Esmon, 1989). For instance,endothelial cell surface thrombomodulin expression and message are knownto be reduced by exposure of the cells to TNF (Conway and Rosenberg,1988; Lentz et al., 1991). To determine if a similar process occurs withEPCR, HUVEC were treated with TNF and APC binding and expression of EPCRmRNA were examined. APC binding to HUVEC decreased in a time dependentfashion. EPCR activity decreased more rapidly than thrombomodulinantigen. HUVEC were cultured for 0, 6, 24 and 48 hr, in the presence ofTNF-α (10 ng/ml). Cells were harvested and residual F1-APC binding orthrombomodulin (TM) expression was analyzed by flow cytometry. Cellsurface TM was stained with an anti-TM murine monoclonal antibody andFITC-conjugated anti-mouse IgG. The negative control is without addedfluorescent ligand.

HUVEC were treated with 10 ng/ml of TNF-α for 0, 0.5, 1, 2, 3, 6, 10 and24 hr, and message was extracted and detected as described above. Theresults demonstrated that the concentration of EPCR mRNA was alsoreduced by TNF treatment. Message levels and APC binding activitydecreased in parallel. Therefore, the TNF mediated down-regulation ofAPC binding to endothelium probably occurs at the level of mRNAexpression.

Enhancement of inflammatory responses by blocking binding of endogenousmolecules to ECPCR can be achieved by administration of compoundsbinding to the receptor to a subject in need of inhibition. The degreeof binding is routinely determined using assays such as those describedabove. Compounds which are effective include antibodies to the protein,fragments of antibodies retaining the binding regions, and peptidefragments of APC which include the Gla region. Inhibition of theinflammatory response could be obtained by infusing soluble EPCR.Alternatively, localizing EPCR to surfaces in contact with blood wouldrender the surfaces anticoagulant by virtue of the ability of EPCR tobind and concentrate the anticoagulant APC at the surface.Alternatively, the function of EPCR could be enhanced by overexpressingthe EPCR in endothelium used to coat vascular grafts in patients withvascular disease or on stents in cardiac patients.

The DNA sequence can also be used for screening for other homologous orstructurally similar receptor proteins using hybridization probes.

These methods and reagents and pharmaceuticals are more readilyunderstood by reference to the following.

Screening of Patient Samples for Expression of Receptor Proteins.

Patients with thrombosis or hyperinflammatory conditions could bescreened for defects in the EPCR gene. Sequence ID No. 1, andconsecutive portions thereof of at least about seven nucleotides, morepreferably fourteen to seventeen nucleotides, most preferably abouttwenty nucleotides, are useful in this screening using hybridizationassays of patient samples, including blood and tissues. Screening canalso be accomplished using antibodies, typically labelled with afluorescent, radiolabelled, or enzymatic label, or by isolation oftarget cells and screening for binding activity, as described in theexamples above. Typically, one would screen for expression on either aqualitative or quantitative basis, and for expression of functionalreceptor. Labelling can be with ³²P, ³⁵S, fluorescein, biotin, or otherlabels routinely used with methods known to those skilled in the art forlabelling of proteins and/or nucleic acid sequences.

Hybridization Probes

Reaction conditions for hybridization of an oligonucleotide probe orprimer to a nucleic acid sequence vary from oligonucleotide tooligonucleotide, depending on factors such as oligonucleotide length,the number of G and C nucleotides, and the composition of the bufferutilized in the hybridization reaction. Moderately stringenthybridization conditions are generally understood by those skilled inthe art as conditions approximately 25° C. below the melting temperatureof a perfectly base-paired double-stranded DNA. Higher specificity isgenerally achieved by employing incubation conditions having highertemperatures, in other words, more stringent conditions. In general, thelonger the sequence or higher the G and C content, the higher thetemperature and/or salt concentration required. Chapter 11 of thewell-known laboratory manual of Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, second edition, Cold Spring Harbor Laboratory Press,New York (1990) (which is incorporated by reference herein), describeshybridization conditions for oligonucleotide probes and primers in greatdetail, including a description of the factors involved and the level ofstringency necessary to guarantee hybridization with specificity.

The preferred size of a hybridization probe is from 10 nucleotides to100,000 nucleotides in length. Below 10 nucleotides, hybridized systemsare not stable and will begin to denature above 20° C. Above 100,000nucleotides, one finds that hybridization (renaturation) becomes a muchslower and incomplete process, as described in greater detail in thetext MOLECULAR GENETICS, Stent, G. S. and R. Calender, pp. 213–219(1971). Ideally, the probe should be from 20 to 10,000 nucleotides.Smaller nucleotide sequences (20–100) lend themselves to production byautomated organic synthetic techniques. Sequences from 100–10,000nucleotides can be obtained from appropriate restriction endonucleasetreatments. The labeling of the smaller probes with the relatively bulkychemiluminescent moieties may in some cases interfere with thehybridization process.

Generation of Antibodies for Diagnostic or Therapeutic Use

Antibodies to the receptor proteins can also be generated which areuseful in detection, characterization or isolation of receptor proteins,as well as for modifying receptor protein activity, in most cases,through inhibition of binding. Antibodies are generated by standardtechniques, using human or animal receptor proteins. Since the proteinsexhibit high evolutionary conservation, it may be advantageous togenerate antibodies to a protein of a different species of origin thanthe species in which the antibodies are to be tested or utilized,looking for those antibodies which are immunoreactive with the mostevolutionarily conserved regions. Antibodies are typically generated byimmunization of an animal using an adjuvant such as Freund's adjuvant incombination with an immunogenic amount of the protein administered overa period of weeks in two to three week intervals, then isolated from theserum, or used to make hybridomas which express the antibodies inculture. Because the methods for immunizing animals yield antibody whichis not of human origin, the antibodies could elicit an adverse effect ifadministered to humans. Methods for “humanizing” antibodies, orgenerating less immunogenic fragments of non-human antibodies, are wellknown. A humanized antibody is one in which only the antigen-recognizedsites, or complementarily-determining hypervariable regions (CDRs) areof non-human origin, whereas all framework regions (FR) of variabledomains are products of human genes. These “humanized” antibodiespresent a lesser xenographic rejection stimulus when introduced to ahuman recipient.

To accomplish humanization of a selected mouse monoclonal antibody, theCDR grafting method described by Daugherty, et al., (1991) Nucl. AcidsRes., 19:2471–2476, incorporated herein by reference, may be used.Briefly, the variable region DNA of a selected animal recombinantanti-idiotypic ScFv is sequenced by the method of Clackson, T., et al.,(1991) Nature, 352:624–688, incorporated herein by reference. Using thissequence, animal CDRs are distinguished from animal framework regions(FR) based on locations of the CDRs in known sequences of animalvariable genes. Kabat, H. A., et al., Sequences of Proteins ofImmunological Interest, 4th Ed. (U.S. Dept. Health and Human Services,Bethesda, Md., 1987). Once the animal CDRs and FR are identified, theCDRs are grafted onto human heavy chain variable region framework by theuse of synthetic oligonucleotides and polymerase chain reaction (PCR)recombination. Codons for the animal heavy chain CDRs, as well as theavailable human heavy chain variable region framework, are built in four(each 100 bases long) oligonucleotides. Using PCR, a grafted DNAsequence of 400 bases is formed that encodes for the recombinant animalCDR/human heavy chain FR protection.

The immunogenic stimulus presented by the monoclonal antibodies soproduced may be further decreased by the use of Pharmacia's (PharmaciaLKB Biotechnology, Sweden) “Recombinant Phage Antibody System” (RPAS),which generates a single-chain Fv fragment (ScFv) which incorporates thecomplete antigen-binding domain of the antibody. In the RPAS, antibodyvariable heavy and light chain genes are separately amplified from thehybridoma mRNA and cloned into an expression vector. The heavy and lightchain domains are co-expressed on the same polypeptide chain afterjoining with a short linker DNA which codes for a flexible peptide. Thisassembly generates a single-chain Fv fragment (ScFv) which incorporatesthe complete antigen-binding domain of the antibody. Compared to theintact monoclonal antibody, the recombinant ScFv includes a considerablylower number of epitopes, and thereby presents a much weaker immunogenicstimulus when injected into humans.

The antibodies can be formulated in standard pharmaceutical carriers foradministration to patients in need thereof. These include saline,phosphate buffered saline, and other aqueous carriers, and liposomes,polymeric microspheres and other controlled release delivery devices, asare well known in the art. The antibodies can also be administered withadjuvant, such as muramyl dipeptide or other materials approved for usein humans (Freund's adjuvant can be used for administration of antibodyto animals).

Screening for Drugs Modifying or Altering the Extent of ReceptorFunction or Expression

The receptor proteins are useful as targets for compounds which turn on,or off, or otherwise regulate binding to these receptors. The assaysdescribed above clearly provide routine methodology by which a compoundcan be tested for an inhibitory effect on binding of PC or APC. The invitro studies of compounds which appear to inhibit binding selectivelyto the receptors are then confirmed by animal testing. Since themolecules are so highly evolutionarily conserved, it is possible toconduct studies in laboratory animals such as mice to predict theeffects in humans.

In cases where inflammatory mediators or vascular disease down regulateEPCR, it would be advantageous to increase its concentration in vivo onendothelium. The binding assays described here and the gene sequenceallow assays for increased EPCR expression. Similar approaches have beentaken with thrombomodulin and investigators have shown that cyclic AMP(Maruyama, I. et al. (1991) Thrombosis Research 61, 301–310) andinterleukin 4 (Kapiotis, S. et al., (1991) Blood 78, 410–415) canelevate thrombomodulin expression. The ability to screen such drugs ordrugs that block TNF down regulation of EPCR provide an approach toelevating EPCR expression in vivo and thus enhancing anticoagulant andanti-inflammatory activity.

Studies based on inhibition of binding are predictive for indirecteffects of alteration of receptor binding. For example, inhibition ofbinding of APC or increased expression of TNF is predictive ofinhibition of EPCR function.

Assays for testing compounds for useful activity can be based solely oninteraction with the receptor protein, preferably expressed on thesurface of transfected cells such as those described above. Proteins insolution or immobilized on inert substrates can also be utilized. Thesecan be used to detect inhibition or enhancement in binding of PC or APC

Alternatively, the assays can be based on interaction with the genesequence encoding the receptor protein, preferably the regulatorysequences directing expression of the receptor protein. For example,antisense which binds to the regulatory sequences, and/or to the proteinencoding sequences can be synthesized using standard oligonucleotidesynthetic chemistry. The antisense can be stabilized for pharmaceuticaluse using standard methodology (encapsulation in a liposome ormicrosphere; introduction of modified nucleotides that are resistant todegradation or groups which increase resistance to endonucleases, suchas phosphorothiodates and methylation), then screened initially foralteration of receptor activity in transfected or naturally occurringcells which express the receptor, then in vivo in laboratory animals.Typically, the antisense would inhibit expression. However, sequenceswhich block those sequences which “turn off” synthesis can also betargeted.

The receptor protein for study can be isolated from either naturallyoccurring cells or cells which have been genetically engineered toexpress the receptor, as described in the examples above. In thepreferred embodiment, the cells would have been engineered using theintact gene.

Random Generation of Receptor or Receptor Encoding Sequence BindingMolecules.

Molecules with a given function, catalytic or ligand-binding, can beselected for from a complex mixture of random molecules in what has beenreferred to as “in vitro genetics” (Szostak, (1992) TIBS 19:89). Onesynthesizes a large pool of molecules bearing random and definedsequences and subjects that complex mixture, for example, approximately10¹⁵ individual sequences in 100 μg of a 100 nucleotide RNA, to someselection and enrichment process. For example, by repeated cycles ofaffinity chromatography and PCR amplification of the molecules bound tothe ligand on the column, Ellington and Szostak (1990) estimated that 1in 10¹⁰ RNA molecules folded in such a way as to bind a given ligand.DNA molecules with such ligand-binding behavior have been isolated(Ellington and Szostak, 1992; Bock et al, 1992).

Computer Assisted Drug Design

Computer modeling technology allows visualization of thethree-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the molecule.The three-dimensional construct typically depends on data from x-raycrystallographic analyses or NMR imaging of the selected molecule. Themolecular dynamics require force field data. The computer graphicssystems enable prediction of how a new compound will link to the targetmolecule and allow experimental manipulation of the structures of thecompound and target molecule to perfect binding specificity. Predictionof what the molecule-compound interaction will be when small changes aremade in one or both requires molecular mechanics software andcomputationally intensive computers, usually coupled with user-friendly,menu-driven interfaces between the molecular design program and theuser.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modeling of drugs interactive withspecific proteins, such as Rotivinen, et al., (1988) Acta PharmaceuticaFennica 97, 159–166; Ripka, New Scientist 54–57 (Jun. 16, 1988);McKinaly and Rossmann, (1989) Annu. Rev. Pharmacol. Toxiciol. 29,111–122; Perry and Davies, OSAR: Quantitative Structure-ActivityRelationships in Drug Design pp. 189–193 (Alan R. Liss, Inc. 1989);Lewis and Dean, (1989) Proc. R. Soc. Lond. 236, 125–140 and 141–162;and, with respect to a model receptor for nucleic acid components,Askew, et al., (1989) J. Am. Chem. Soc. 111, 1082–1090. Other computerprograms that screen and graphically depict chemicals are available fromcompanies such as BioDesign, Inc., Pasadena, Calif., Allelix, Inc,Mississauga, Ontario, Canada, and Hypercube, Inc., Cambridge, Ontario.Although these are primarily designed for application to drugs specificto particular proteins, they can be adapted to design of drugs specificto regions of DNA or RNA, once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Generation of Nucleic Acid Regulators

Nucleic acid molecules containing the 5′ regulatory sequences of thereceptor genes can be used to regulate or inhibit gene expression invivo. Vectors, including both plasmid and eukaryotic viral vectors, maybe used to express a particular recombinant 5′ flanking region-geneconstruct in cells depending on the preference and judgment of theskilled practitioner (see, e.g., Sambrook et al., Chapter 16).Furthermore, a number of viral and nonviral vectors are being developedthat enable the introduction of nucleic acid sequences in vivo (see,e.g., Mulligan, (1993) Science, 260, 926–932; U.S. Pat. No. 4,980,286;U.S. Pat. No. 4,868,116; incorporated herein by reference). Recently, adelivery system was developed in which nucleic acid is encapsulated incationic liposomes which can be injected intravenously into a mammal.This system has been used to introduce DNA into the cells of multipletissues of adult mice, including endothelium and bone marrow (see, e.g.,Zhu et al., (1993) Science 261, 209–211; incorporated herein byreference).

The 5′ flanking sequences of the receptor gene can also be used toinhibit the expression of the receptor. For example, an antisense RNA ofall or a portion of the 5′ flanking region of the receptor gene can beused to inhibit expression of the receptor in vivo. Expression vectors(e.g., retroviral expression vectors) are already available in the artwhich can be used to generate an antisense RNA of a selected DNAsequence which is expressed in a cell (see, e.g., U.S. Pat. No.4,868,116; U.S. Pat. No. 4,980,286). Accordingly, DNA containing all ora portion of the sequence of the 5′ flanking region of the receptor genecan be inserted into an appropriate expression vector so that uponpassage into the cell, the transcription of the inserted DNA yields anantisense RNA that is complementary to the mRNA transcript of thereceptor protein gene normally found in the cell. This antisense RNAtranscript of the inserted DNA can then base-pair with the normal mRNAtranscript found in the cell and thereby prevent the mRNA from beingtranslated. It is of course necessary to select sequences of the 5′flanking region that are downstream from the transcriptional start sitesfor the receptor protein gene to ensure that the antisense RNA containscomplementary sequences present on the mRNA.

Antisense RNA can be generated in vitro also, and then inserted intocells. Oligonucleotides can be synthesized on an automated synthesizer(e.g., Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). In addition, antisensedeoxyoligonucleotides have been shown to be effective in inhibiting genetranscription and viral replication (see e.g., Zamecnik et al., (1978)Proc. Natl. Acad. Sci. USA 75, 280–284; Zamecnik et al., (1986) Proc.Natl. Acad. Sci., 83, 4143–4146; Wickstrom et al., (1988) Proc. Natl.Acad. Sci. USA 85, 1028–1032; Crooke, (1993) FASEB J. 7, 533–539.Furthermore, recent work has shown that improved inhibition ofexpression of a gene by antisense oligonucleotides is possible if theantisense oligonucleotides contain modified nucleotides (see, e.g.,Offensperger et. al., (1993) EMBO J. 12, 1257–1262 (in vivo inhibitionof duck hepatitis B viral replication and gene expression by antisensephosphorothioate oligodeoxynucleotides); PCT WO 93/01286 Rosenberg etal., (synthesis of sulfurthioate oligonucleotides); Agrawal et al.,(1988) Proc. Natl. Acad. Sci. USA 85, 7079–7083 (synthesis of antisenseoligonucleoside phosphoramidates and phosphorothioates to inhibitreplication of human immunodeficiency virus-1); Sarin et al., (1989)Proc. Natl. Acad. Sci. USA 85, 7448–7794 (synthesis of antisensemethylphosphonate oligonucleotides); Shaw et al., (1991) Nucleic AcidsRes 19, 747–750 (synthesis of 3′ exonuclease-resistant oligonucleotidescontaining 3′ terminal phosphoroamidate modifications); incorporatedherein by reference).

The sequences of the 5′ flanking region of receptor protein gene canalso be used in triple helix (triplex) gene therapy. Oligonucleotidescomplementary to gene promoter sequences on one of the strands of theDNA have been shown to bind promoter and regulatory sequences to formlocal triple nucleic acid helices which block transcription of the gene(see, e.g., Maher et al., (1989) Science 245, 725–730; Orson et al.,(1991) Nucl. Acids Res. 19, 3435–3441; Postal et al., (1991) Proc. Natl.Acad. Sci. USA 88, 8227–8231; Cooney et al., (1988) Science 241,456–459; Young et al., (1991) Proc. Natl. Acad. Sci. USA 88,10023–10026; Duval-Valentin et al., (1992) Proc. Natl. Acad. Sci. USA89, 504–508; Blume et al., (1992) Nucl. Acids Res. 20, 1777–1784;Grigoriev et al., (1992) J. Biol. Chem. 267, 3389–3395.

Recently, both theoretical calculations and empirical findings have beenreported which provide guidance for the design of oligonucleotides foruse in oligonucleotide-directed triple helix formation to inhibit geneexpression. For example, oligonucleotides should generally be greaterthan 14 nucleotides in length to ensure target sequence specificity(see, e.g., Maher et al., (1989); Grigoriev et al., (1992)). Also, manycells avidly take up oligonucleotides that are less than 50 nucleotidesin length (see e.g., Orson et al., (1991); Holt et al., (1988) Mol.Cell. Biol. 8, 963–973; Wickstrom et al., (1988) Proc. Natl. Acad. Sci.USA 85, 1028–1032). To reduce susceptibility to intracellulardegradation, for example by 3′ exonucleases, a free amine can beintroduced to a 3′ terminal hydroxyl group of oligonucleotides withoutloss of sequence binding specificity (Orson et al., 1991). Furthermore,more stable triplexes are formed if any cytosines that may be present inthe oligonucleotide are methylated, and also if an intercalating agent,such as an acridine derivative, is covalently attached to a 5′ terminalphosphate (e.g., via a pentamethylene bridge); again without loss ofsequence specificity (Maher et al., (1989); Grigoriev et al., (1992).

Methods to produce or synthesize-oligonucleotides are well known in theart. Such methods can range from standard enzymatic digestion followedby nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5,6) to purely synthetic methods, for example, by the cyanoethylphosphoramidite method using a Milligen or Beckman System 1Plus DNAsynthesizer (see also, Ikuta et al., (1984) Ann. Rev. Biochem. 53,323–356 (phosphotriester and phosphite-triester methods); Narang et al.,(1980) Methods Enzymol., 65, 610–620 (phosphotriester method).Accordingly, DNA sequences of the 5′ flanking region of the receptorprotein gene described herein can be used to design and constructoligonucleotides including a DNA sequence consisting essentially of atleast 15 consecutive nucleotides, with or without base modifications orintercalating agent derivatives, for use in forming triple helicesspecifically within the 5′ flanking region of a receptor protein gene inorder to inhibit expression of the gene.

In some cases it may be advantageous to insert enhancers or multiplecopies of the regulatory sequences into an expression system tofacilitate screening of methods and reagents for manipulation ofexpression.

Preparation of Receptor Protein Fragments

Compounds which are effective for blocking binding of the receptor canalso consist of fragments of the receptor proteins, expressedrecombinantly and cleaved by enzymatic digest or expressed from asequence encoding a peptide of less than the full length receptorprotein. These will typically be soluble proteins, i.e., not includingthe transmembrane and cytoplasmic regions, although smaller portionsdetermined in the assays described above to inhibit or compete forbinding to the receptor proteins can also be utilized. It is a routinematter to make appropriate receptor protein fragments, test for binding,and then utilize. The preferred fragments are of human origin, in orderto minimize potential immunological response. The peptides can be asshort as five to eight amino acids in length and are easily prepared bystandard techniques. They can also be modified to increase in vivohalf-life, by chemical modification of the amino acids or by attachmentto a carrier molecule or inert substrate. Based on studies with otherpeptide fragments blocking receptor binding, the IC₅₀, the dose ofpeptide required to inhibit binding by 50%, ranges from about 1 μM togreater than 10 mM, depending on the peptide size and folding. Theseranges are well within the effective concentrations for the in vivoadministration of peptides, based on comparison with the RGD-containingpeptides, described, for example, in U.S. Pat. No. 4,792,525 toRuoslaghti, et al., used in vivo to alter cell attachment andphagocytosis. The peptides can also be conjugated to a carrier proteinsuch as keyhole limpet hemocyanin by its N-terminal cysteine by standardprocedures such as the commercial Imject kit from Pierce Chemicals orexpressed as a fusion protein, which may have increased efficacy.

As noted above, the peptides can be prepared by proteolytic cleavage ofthe receptor proteins, or, preferably, by synthetic means. These methodsare known to those skilled in the art. An example is the solid phasesynthesis described by J. Merrifield, (1964) J. Am. Chem. Soc. 85, 2149,used in U.S. Pat. No. 4,792,525, and described in U.S. Pat. No.4,244,946, wherein a protected alpha-amino acid is coupled to a suitableresin, to initiate synthesis of a peptide starting from the C-terminusof the peptide. Other methods of synthesis are described in U.S. Pat.Nos. 4,305,872 and 4,316,891. These methods can be used to synthesizepeptides having identical sequence to the receptor proteins describedherein, or substitutions or additions of amino acids, which can bescreened for activity as described above.

The peptide can also be administered as a pharmaceutically acceptableacid- or base-addition salt, formed by reaction with inorganic acidssuch as hydrochloric acid, hydrobromic acid, perchloric acid, nitricacid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organicacids such as formic acid, acetic acid, propionic acid, glycolic acid,lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,maleic acid, and fumaric acid, or by reaction with an inorganic basesuch as sodium hydroxide, ammonium hydroxide, potassium hydroxide, andorganic bases such as mono-, di-, trialkyl and aryl amines andsubstituted ethanolamines.

Peptides containing cyclopropyl amino acids, or amino acids derivatizedin a similar fashion, can also be used. These peptides retain theiroriginal activity but have increased half-lives in vivo. Methods knownfor modifying amino acids, and their use, are known to those skilled inthe art, for example, as described in U.S. Pat. No. 4,629,784 toStammer.

The peptides are generally active when administered parenterally inamounts above about 1 μg/kg of body weight. Based on extrapolation fromother proteins, for treatment of most inflammatory disorders, the dosagerange will be between 0.1 to 70 mg/kg of body weight. This dosage willbe dependent, in part, on whether one or more peptides are administered.

Pharmaceutical Compositions

Compounds which alter receptor protein binding are preferablyadministered in a pharmaceutically acceptable vehicle. Suitablepharmaceutical vehicles are known to those skilled in the art. Forparenteral administration, the compound will usually be dissolved orsuspended in sterile water or saline. For enteral administration, thecompound will be incorporated into an inert carrier in tablet, liquid,or capsular form. Suitable carriers may be starches or sugars andinclude lubricants, flavorings, binders, and other materials of the samenature. The compounds can also be administered locally by topicalapplication of a solution, cream, gel, or polymeric material (forexample, a Pluronic™, BASF).

Alternatively, the compound may be administered in liposomes ormicrospheres (or microparticles). Methods for preparing liposomes andmicrospheres for administration to a patient are known to those skilledin the art. U.S. Pat. No. 4,789,734 describe methods for encapsulatingbiological materials in liposomes. Essentially, the material isdissolved in an aqueous solution, the appropriate phospholipids andlipids added, along with surfactants if required, and the materialdialyzed or sonicated, as necessary. A review of known methods is by G.Gregoriadis, Chapter 14. “Liposomes”, Drug Carriers in Biology andMedicine pp. 287–341 (Academic Press, 1979). Microspheres formed ofpolymers or proteins are well known to those skilled in the art, and canbe tailored for passage through the gastrointestinal tract directly intothe bloodstream. Alternatively, the compound can be incorporated and themicrospheres, or composite of microspheres, implanted for slow releaseover a period of time, ranging from days to months. See, for example,U.S. Pat. Nos. 4,906,474, 4,925,673, and 3,625,214.

Disorders to be Treated

As described herein, a variety of compounds can be used to inhibit orenhance expression of the EPCR. The nature of the disorder willdetermine if the expression should be enhanced or inhibited. Forexample, based on the studies involving the use of an anti-protein Cantibody in combination with cytokine, it should be possible to treatsolid tumors by enhancing an inflammatory response involving blocking ofprotein C or activated protein C binding to an endothelial cell proteinC/activated protein C receptor by administering to a patient in need oftreatment thereof an amount of a compound blocking binding of protein Cor activated protein C to the receptor. Similarly, it should be possibleto treat disorders such as gram negative sepsis, stroke, thrombosis,septic shock, adult respiratory distress syndrome, and pulmonary emboliusing a method for inhibiting an inflammatory response involvingadministration of EPCR or EPCR fragments or substances that upregulateEPCR expression to a patient in need of treatment therof.

Modifications and variations of the methods and materials describedherein will be obvious to those skilled in the art and are intended tobe encompassed by the following claims. The teachings of the referencescited herein are specifically incorporated herein.

1. A method for enhancing an inflammatory response involving blocking ofprotein C or activated protein C binding to an endothelial cell proteinC/activated protein C receptor comprising administering to a patient inneed of treatment thereof an antibody or antibody fragmentimmunoreactive with said endothelial cell protein C/activated protein Creceptor in an amount sufficient to block binding of protein C oractivated protein C to the receptor by binding to the endothelial cellprotein C/activated protein C receptor.
 2. The method of claim 1 whereinthe antibody is humanized.
 3. The method of claim 1 wherein the antibodyis labeled.
 4. The method of claim 1 wherein the antibody is combinedwith a pharmaceutically acceptable carrier.